본초학표지

大韓本草學會誌제 28 권제 4 호 (2013 년 7 월 ) Kor. J. Herbology 2013;28(4):57-62 ISSN 1229-1765 http://dx.doi.org/10.6116/kjh.2013.28.4.57. Hydrogen peroxide로산화적스트레스가유도된 HaCaT keratinocyte 에서금은화의세포보호효과 서승희 #, 최미옥 * 광주여자대학교미용과학과 Protectvie effects of Lonicerae Japonicae Flos against hydrogen peroxidase-induced oxidative stress on Human keratinocyte, HaCaT cells. Seung-Hee Seo #, Mee-Ok Choi * Dept. of Beauty Science Graduate School, Kwangju Women's University. ABSTRACT Objectives : Lonicerae Japonicae Flos (LJF) has been shown anti-oxidant, anti-inflammatory, anti-viral, anti-rheumatoid properties. However, it is still largely unknown whether LJF inhibits skin injury against oxidative stress in human keratinocyte, HaCaT cells. The purpose of this study was to evaluate the protective effects of LJF against hydrogen peroxide(h 2O 2)-induced oxidative stress in human keratinocytes, HaCaT cells. Methods : To evaluate out the protective effects of LJF on oxidative injury in HaCaT cells, an oxidative stress model of HaCaT cells was established under a suitable concentration (500 µm) hydrogen peroxide. HaCaT keratinocyte cells were pre-treated with LJF (0.1, 0.25 or 0.5 mg/ml), and then stimulated with H 2O 2. Then, the cells were harvested to measure the cell viability, DNA damage, and release of reactive oxygen species (ROS). Results : LJF (0.1, 0.25 or 0.5 mg/ml) itself did not show any significant toxicity in HaCaT cells. The treatment of H 2O 2 caused the oxidative stress, leading to the cell death, and DNA injury. However, pretreatment with LJF reduced cell death, and DNA injury. The stimulation of H 2O 2 on HaCaT cells resulted in excessive release of ROS, which is the main factor of oxidative stress. The excessive release of ROS was inhibited by LJF treatment significantly. Conclusions : These results could suggest that LJF exhibited the protective effects of HaCaT cells against H 2O 2-induced oxidative stress by inhibiting ROS release. It could be explained that LJF inhibit skin damages against oxidative stress. Thus, LJF would be useful for the development of drug or cosmetics treating skin troubles. Key words : Keratinocyte, HaCaT cell, Lonicerae Japonicae Flos, hydrogen peroxide (H 2O 2), reactive oxygen species (ROS) 서론 1) 금은화 ( 金銀花, Lonicera japonica Flos, LJF) 의기원식물은대한약전과중국약전에의하면다양하다. 대한약전에서는인동과 (Caprifoliaceae) 에속한인동덩굴 Lonicera japonica Thunb. 의꽃봉오리를기원으로하지만중국약전에서는인동덩굴은물론, 화남인동 ( 華南忍冬 ) Lonicera confusa (Sweet) DC, 고선인동 ( 菰腺忍冬 ) Lonicera hypoglauca Miq. 황갈모인동 ( 黄褐毛忍冬 ) Lonicera fulvotomentosa * 교신저자 : 최미옥, 광주광역시광산구여대길 201 번지광주여자대학교미용과학과 Tel : 063-850-6837 E-mail : cmo0323@hanmail.net # 제 1 저자 : 서승희, 광주광역시광산구여대길 201 번지광주여자대학교미용과학과 Tel : 063-850-6837 E-mail : joahaeu@naver.com 접수 :2013 년 6 월 19 일 수정 :2013 년 7 월 3 일 채택 :2013 년 7 월 19 일

58 大韓本草學會誌 Vol. 28 No. 4, 2013 Hsu et S. C. Cheng 등을기원으로사용한다. 본연구에서는인동덩굴 Lonicera japonica Thunb. 을이용하여실험을진행하였다. 한방에서는性味가寒하고甘하며, 心, 肺, 胃經에歸經하여涼散風熱, 擁腫精瘡, 喉痹, 丹毒, 熱血毒痢, 風血感冒, 溫炳發熱의效能이있어서癰腫疔瘡, 喉痺, 丹毒, 熱毒血痢, 風熱感冒, 溫病發熱等의證狀에常用되고있다 1,2). 또한, 임상적으로감염성질환, 외이도염, 화농증, 중이염과질환뿐만아니라바이러스성결막염, 인플루엔자, 폐렴등의염증성질병에도사용되고있다 3,4). 이외의알려진효능으로 in vitro 모델에서의연구에서항균, 항바이러스작용으로그람양성균과그람음성균에대해억제작용이있으며 5), 항산화작용 6), 간보호작용 7), 항암작용 8), 면역증강 9) 등이연구보고되어있다. 인체피부는크게표피, 진피, 피하조직으로구성되어있다. 이중에서각질형성세포 (keratinocytes) 는표피층에존재하고있으며, 외부환경으로부터신체내부를보호하거나보습, 체온조절등중요한역할을한다 10). 하지만피부표피에해당하는각질형성세포 (keratinocytes) 는바깥층에서태양광선 ( 자외선 ) 을비롯한외부자극에직접적으로노출되게되는데이러한지속적인외부자극의노출을통해산화적스트레스 (oxidative stress) 가발생하게된다. 산화적스트레스 (oxidative stress) 는과산화수소 (hydrogen peroxide, H 2O 2), 수산기 (hydroxyl radicals, OH - ), 페록실기 (peroxyl radicals, ROO - ), 퍼옥시니트라이트 (peroxynitrite, ONOO - ) 그리고과산화물음이온 (superoxide anion, O - 2 ) 을비롯한다양한활성산소 (reactive oxygen species, ROS) 를생성하여각질형성세포의탄력및보습기능의저하, 노화의촉진, 그리고피부세포의염증과괴사등을유발하는것으로알려졌다 11,12). 따라서산화적스트레스 (oxidative stress) 로부터각질형성세포를보호하고노화를지연시키기위해서는체내항산화방어시스템을향상시키는것이매우중요하다 13). 선행연구들에서금은화의항산화, 항염증과같은다양한연구가진행되었으나, 피부에서금은화의세부적인효능연구는보고된바가없었다. 이에본연구에서는각질형성세포 HaCaT keratinocyte 에서과산화수소 (hydrogen peroxide, H 2O 2) 로유발되는산화적스트레스에대한금은화물추출물의효과를알아보고자하였다. 1. 재료 1) 약재 재료및방법 실험에사용한금은화 (Lonicerae Japonicae Flos) 는옴니허브 ( 경북영천, 대한민국 ) 에서구입하여정선해서사용하였다. 2) 세포주및세포배양본실험에사용된 HaCaT 세포는 Addexobio (San Diego, Califonia, USA) 로부터분양받아실험에사용하였다. HaCaT 세포는형질전환된각질형성세포로사람피부의정상각질형성세포와형태및반응양식이동일하면서계대배양이제한되 지않아유지하기가편한장점이있다. 각질형성세포주인 HaCaT 세포주를 10% fetal bovine serum(fbs) 과 1% penicillin/streptomycine 을첨가한 Dulbecco s modified Eagle s medium (DMEM) (GibcoBRL, Braunscheig, Germany) 배지를사용하여 5% CO 2, 37 세포배양기에서배양하면서실험에사용하였다. 2. 방법 1) 약물추출금은화 100g을 1L에약탕기 ( 대웅, 한국 ) 로 3시간가열추출한다음여과한후, 동결건조하여건조분말 (15.3g) 을얻었으며실험을위하여 4 에보관했다 ( 수율 :15.3%). 생리식염수녹여 stock solution (100 mg/ml) 을제조하였다. 제조된 stock solution 은 -20 에보관하면서분석방법에적합하도록희석하여사용하였다. 2) 세포독성분석 HaCaT cell의생존율은밀집세포의미토콘드리아탈수소효소에의해자줏빛 formazan 생성물로변하는 MTT 환원을바탕으로 MTT 분석법으로측정했다. 세포들은 DMEM 배지에서 5 10 4 cells/well 의밀도로현탁하여 0.1, 0.25, 0.5 mg/ml의농도로금은화물추출물을처리하였다. 24시간배양후 5 mg/ml 의농도로 MTT 용액을첨가하고다시 30 분동안배양하였다. MTT-formazan 생성물은 DMSO 200 µl를첨가함으로써용해했다. formazan의양은용해액을 96well plate에 loading 한후, 540 nm에흡수되는양을측정함으로서결정하였다. 3) DNA 응축분석 (4,6-diamidino-2-phenylindole, DAPI) DAPI (Sigma, MO, USA) 는 intact cell membrane을투과하여 DNA와결합하게되고, 이때 358nm(Max.) 정도파장대의빛을흡수하여 blue-cyan 계통의형광을발한다. 그렇게결합한 DAPI는 DNA를염색함으로서, 핵과 chromatin 의형태를관찰할수있게해준다. HaCaT 세포에 H2O2 를처리한후 1시간동안배양하였다. PBS로 2회세척후 4% paraformaldehyde 용액으로 30분동안실온에서고정하고 PBS로 3회세척하였다. 세포의핵은 DAPI (10 mg/ml) 사용하여 5분간염색하고 PBS로 3회세척하였고, prolong gold anti-fading mount solution (invitrogen, CA, USA) 를이용하여마운팅하였다. 그후 Flourscence microscopy (Olympus 70, Tokyo, Japan) 으로관찰하였다. 4) DNA 분절측정 (1) 세포 DNA분절샘플준비 Cellular DNA fragmentation Kit (Millipore, MA, USA) 를이용하여 DNA의분절정도를측정하였다. 먼저 HaCaT 세포를 2 10 5 cells/ml로현탁하고, bradu labeling solution 을 10 µm을첨가하여 5% CO 2, 37 조건하에서 2시간배양하였다. 그후 250 g에서 10분간원심분리하여상층액을제거한다음, 세포에 DMEM 배지를각 200 µl씩첨가하여 96well plate에넣어주었다. 실험군에 LJ를 0.1, 0.25, 0.5

Hydrogen peroxide 로산화적스트레스가유도된 HaCaT keratinocyte 에서금은화의세포보호효과 59 mg/ml 농도로한후 1시간뒤, H 2O 2 500 µm을처리하고 5% CO 2, 37 조건하에서 1시간배양하였다. 1시간후에 96well plate를 250 g에서 10분간원심분리후, 상층액을제거하였다. 96well plate 각 well에 1 incubation solution 200 µl를첨가한후, 상온에서 30분간배양하였다. 그후 250 g에서 10분간원심분리하여나온상층액을 100 µl 취하여실험에사용하였다. 처리하여 3시간동안배양한후세포독성분석법을실시해생존율을관찰하였다. 그결과, 아래 Fig. 1과같이금은화는 HaCaT 세포에서최고농도 0.5 mg/ml까지는유의성있는세포독성을보이지않았으나, 1 mg/ml 의농도에서는유의성있는세포독성을보여, 실험의농도를 0.1, 0.25, 0.5 mg/ml로설정하였다 (Fig. 1). (2) 세포 DNA 분절분석 96 well plate에 DNA coating solution 100 µl를첨가한뒤, 4 에서 overnight 하였다. 현탁액을털어내어버린뒤, 1 incubation solution 200 µl를첨가하여상온에서 30분간 blocking 하였다. 그후, washing buffer 200 µl를첨가하여 3회씻어낸다음, exonuclease Ⅲ solution 100 µl를첨가하여 37 에서 30분간 denaturing 하였다. washing buffer 200 µl를첨가하여 3회씻어낸다음, anti-brad-pod conjugate solution을각 well에 100 µl씩첨가하여 4 에서 overnight 하였다. 그후, washing buffer 200 µl를첨가하여 3회씻어낸다음, substrate solution 100 µl를첨가해 DNA가분절된정도를 370 nm에서 spectrometer 를이용하여흡광도를측정함으로서결정하였다. 5) ROS 생성도분석 (Dichlorofluorescein diacetate, DCF-DA) 세포내활성산소의생성을측정하기위하여형광 probe는 DCF-DA 를이용하였다. 비형광물질인 DCF-DA (Sigma, MO, USA) 는세포내 H 2O 2 와관련된 peroxide 존재시 ROS 에의해산화되어진다. 2,7-dichlorofluorescein 로탈에스테르화된 DCF-DA 는형광의 DCF로변환되어녹색의형광을띄게된다. HaCaT 세포에 H 2O 2 를처리한후 1시간동안배양하였다. 세포를수확하기전에 2 µm DCF-DA 를처리하여 37 에서 10분동안배양하였다. 배양한세포는 PBS로세척하여 1% trypsine-edta 용액을처리하여세포를수확하고, 다시 PBS로세척하여 flow cytometry (FACS Calibur, BD Biosciences) 로형광을측정하였다. 정보의분석은 Cell Quest software( Becton Dickinson) 를이용하였다. Fig 1. The effects of Lonicerae Japonicae Flos (LJF) extracts on cell viability in human keratinocyte HaCaT cells. HaCaT cells were incubated with or without LJF indicated doses (0.1, 0.25, 0.5 or 1 mg/ml) for 3 h. The cell viability was measured by MTT assay. The results sere similar in 3 additional experiments. *p < 0.05 significant as compared to untreated normal. 2. 금은화물추출물이 H 2 O 2 로유도한 HaCaT 세포의독성에미치는영향 활성산소의한종류로잘알려진 H 2O 2 는직접적으로사람의각질형성세포내에서산화적스트레스를유발한다고알려져있다 12,13). 따라서, 먼저 H 2O 2 처리후사람각질형성세포인 HaCaT 세포에어떠한영향을미치는지알아보고, 금은화의효과를살펴보고자하였다. HaCaT 세포에금은화를 0.1, 0.25, 0.5 mg/ml 농도별로처리하여 1시간동안배양한후 H 2O 2 500 µm 을 2시간처리한뒤세포생존률을살펴보았다. 그결과 H 2O 2 500 µm을처리하였을때감소하던세포생존률이금은화처리군에서농도의존적으로증가한것을확인할수있었다 (Fig. 2). 6) 통계처리실험결과에대한통계처리는 SPSS V10.0을이용하여 one way ANOVA로검정하여 P값이 0.05 미만일때통계적으로유의한차이가있는것으로판정하였다. 결과 1. 금은화물추출물이 HaCaT 세포독성에미치는영향금은화물추출물이사람각질형성세포인 HaCaT 세포의대사및독성에영향을주어세포사멸및산화적스트레스발생에관여할수있음을배제하기위하여, HaCaT 세포에농도별로금은화를 0.1, 0.25, 0.5, 혹은 1 mg/ml의농도로 Fig 2. The effects of LJF extracts on the H 2O 2-induced cytotoxicity in human keratinocyte HaCaT cells. HaCaT cells were pre-treated with or without LJF (0.1, 0.25, or 0.5 mg/ml) for 1h, then induced with H 2O 2 (500 µm). After 2 h, the cell viability was measured by MTT assay. The results were similar in 3 additional experiments. *p < 0.05 significant as compared to untreated normal, p < 0.05 : significant as compared to H 2O 2 alone. 3. 금은화물추출물이 H 2 O 2 로유도된산화적스 트레스로인한세포내 DNA 손상에미치는영향 H 2O 2 처리시나타나는세포 DNA 손상에금은화가주는영향을알아보기위하여, 금은화를농도별 (0.1, 0.25, 0.5

60 大韓本草學會誌 Vol. 28 No. 4, 2013 mg/ml) 로 1시간전처리하고, H 2O 2 (500 µm) 을 1시간처리하였다. Fig. 3에서와보는바와같이정상군에비하여대조군인 H 2O 2 처리군에서핵의응축이일어나는것을확인할수있었다. 하지만금은화를 0.1, 0.25, 0.5 mg/ml 을처리하였을때 H 2O 2 (500 µm) 처리로인한핵의응축을유의하게감소되는것을볼수있었다 (Fig 3). to untreated normal, p < 0.05 : significant as compared to H 2O 2 alone. 5. 금은화물추출물이 H 2 O 2 로유도된산화적스 트레스로인한 ROS 생성에미치는영향 H 2O 2 처리시 ROS 생성이활성화되고, ROS 분비가사람각질형성세포의사멸및염증반응을촉진한다는보고에따라서 14,15), 금은화가 ROS의생성에어떤영향을주는지알아보기위하여, 금은화를농도별 (0.1, 0.25, 0.5 mg/ml) 로 1시간전에전처리하고, H 2O 2 500 µm을처리하였다. H 2O 2 처리 1시간후세포에 DCF-DA를염색하여, ROS의생성을관찰하였다. 그결과정상군보다 H 2O 2 (500 µm) 처리군에서 ROS 생성이크게증가하였다. 하지만금은화를처리하였을때 H 2O 2 처리로인한 ROS 생성이농도의존적으로유의하게억제됨을확인할수있었다 (Fig. 5). Fig 3. The effects of LJF on the H 2O 2-induced cell death in human keratinocyte HaCaT cells. The cells were pre-treated with or without LJF (0.1, 0.25, or 0.5 mg/ml) for 1h, and then induced with or with out H 2O 2 (500 µm). After 1 h, the cells were harvested for detection of DAPI. To find our the DNA fragment, the cells were stained with DAPI. Detail method were described Materials and Method. The similar results were obtained from three additional experiment. *p < 0.05 significant as compared to untreated normal, p < 0.05 : significant as compared to H 2O 2 alone. 4. 금은화물추출물이 H 2 O 2 로유도된산화적스 트레스로인한 DNA 분절에미치는영향 H 2O 2 처리시나타나는세포 DNA 분절에금은화가주는영향을알아보기위하여, 금은화를 0.1, 0.25, 0.5 mg/ml을 H 2O 2 처리 1시간전에전처리하고, 1시간후 H 2O 2 (500 µm) 을 1시간처리하였다. 그결과정상군에비하여대조군인 H 2O 2 처리군에서 DNA분절이일어나는것을확인할수있었다 (Fig 4). 하지만금은화를 0.1, 0.25, 0.5 mg/ml을처리하였을때 H 2O 2 (500 µm) 처리로인한 DNA 분절을유의하게감소시킬수있었다 (Fig. 4). Fig 4. The effects of LJF on the H 2O 2-induced DNA fragmentation in human keratinocyte HaCaT cells. The cells were pre-treated with or without LJF (0.1, 0.25, or 0.5 mg/ml) for 1h, and then induced with or without H 2O 2 (500 µm). After 1 h, the cells were harvested for detection of DNA fragmentation. The cells were visualized by DNA fragmentation assay. Detail method were described Materials and Method. The similar results were obtained from three additional experiment. *p < 0.05 significant as compared Fig 5. The effects of LJF on the hydrogen peroxide-induced ROS production in human keratinocyte HaCaT cells. The cells were pre-treated with or without LJF (0.1, 0.25, or 0.5 mg/ml) for 1 h, and then induced with or without H 2O 2 (500 µm). After 1 h, the cells were harvested for detection of ROS. Then the ROS production were measured by DCFDA positive cell percent. Detail methods were described Materials and Methods. The similar results were obtained from three additional experiments. *p < 0.05 significant as compared to untreated normal, p < 0.05 : significant as compared to H 2O 2 alone. 고찰 최근삶의질개선과함께피부관련분야에대한관심이높아지면서외부환경적요인에의한피부노화를개선시키기위해화장품, 의약품등을비롯한다양한분야에서항노화, 항산화에대한연구가활발하게이루어지고있다. 자외선을비롯한지속적인외부자극에피부가노출될경우산화적인피부손상은물론광노화를유발한다고알려져있다 16). 외부자극에의한피부노화는피부의각질형성세포와섬유아세포를활성화시켜유해한활성산소 (ROS) 를생성하여산화적스트레스를유발하며결국에는피부세포의손상은물론나아가서피부의퇴화나사멸을초래하게된다고알려져있다 17,18). 각질형성세포라불리는 Keratinocytes 는피부에있어다양한외부환경과자극으로부터피부를보호해주고, 수분등의내부물질이피부밖으로소실되지않도록방어하는중요한역할을한다. 그러므로피부최외곽에서다양한스트레스에노출되는 keratinocytes 에서산화적스트레스에대한방어시스템은중요한의미를가진다. 하지만과도한산화적스트레스에의

Hydrogen peroxide 로산화적스트레스가유도된 HaCaT keratinocyte 에서금은화의세포보호효과 61 해 keratinocyte 가손상되면, 방어시스템의붕괴가와서피부에손상을입게된다. 따라서, 본연구에서는항산화, 항염증에유용하게사용되어온금은화를이용하여 HaCaT keratinocytes 에서항산화활성을평가하고세포손상을억제하는효과를탐색하였다. 금은화는오래전부터한의학에서는호흡기질환, 관절염치료, 해독및해열제로사용되어왔으며 19), 이러한주요효능은금은화에함유되어있는항산화물질에의한것임이밝혀져, 최근에는금은화의함유성분들과그생리활성에대한연구가진행되고있다 20). 또한, 최근진행된연구로는금은화에틸아세테이트분획물의돌연변이생성억제 21) 및항산화효과 22) 에관한내용과금은화주요함유성분들의 HIV-1 RT 저해 23), phospholipase A2 저해 24) 등이보고되었다. 그러나피부세포에서의금은화의효과에관한국내외연구는미미한실정이며, 특히각질형성세포에서의항산화효과에관련된연구는진행된바가없었다. 본실험에서는금은화물추출물의항산화활성을평가하기위해서 HaCaT keratinocytes 에활성산소의한종류로잘알려진 H 2O 2 (500 µm) 를이용하여산화적스트레스를유도하였다. H 2O 2 는산화적스트레스뿐만아니라산소 radical 의모든부분에관여하는것으로, 대부분의세포와조직에자유롭게분포한다 25). 또한 H 2O 2 는다양한신호기전에관여하여생리활성에주요한작용을한다 26,27). 최근많은연구에서는 H 2O 2 를통한세포사멸에초점을맞추고있다 28,29). H2O2는세포사멸과정에서형태학적인세포사멸, 세포수축, DNA 분절등다양한변화를일으킨다 30). 이러한변화에는다양한원인이존재하지만, ROS가주요하게작용한다고알려져있다 31,32). ROS는 DNA의구조를붕괴시키면서 DNA를손상시키고, 세포소기관들을다양하게공격하면서세포사멸및염증을유도하는것으로알려져있다 33-35). 고로 ROS를통한세포손상및사멸을억제하는것은세포보호에있어서아주중요한의미를가진다. 본연구에서는기존의연구와일치하게 H 2O 2 가세포사멸을증가함을알수있었다. 또한 H 2O 2 는 DNA chromatin 응축및 DNA 분절등을유도하여세포손상을유도하였다. 이에금은화물추출물을전처리한후, H 2O 2 를통하여과도한산화적스트레스를주었을때, 이러한세포사멸및손상을농도의존적으로억제함을알수있었다. 또한 H 2O 2 에의한과도한 ROS생성도금은화에의해억제됨을보았을때, 금은화는 H 2O 2 에의한 ROS 생성을억제하여서추가적인세포사멸및손상을억제함을유추할수있다. 본연구에서는금은화물추출물의사람각질형성세포에서의항산화효과를통한세포보호효과를살펴보고자하였으며, 그결과금은화물추출물은각질형성세포에서의항산화, 세포보호효과가우수하였다. 따라서, 본연구결과는추후에금은화를이용한피부에서의다양한효능및기전연구를하는데기초적인자료가될것으로판단된다. 또한, 추후연구에서금은화물추출물의피부에대한세부기전연구및활성성분분석에대한추가적인실험이필요할것으로사료된다. 결론 금은화물추출물을이용하여사람각질형성세포인 HaCaT keratinocyte 에서 H2O2로유발한산화적인손상에대한효과를관찰한결과다음과같은결론을얻었다. 1. 금은화물추출물은 HaCaT keratinocyte 에서 0.5 mg/ml에서까지유의성있는세포독성을보이지않았으며, H 2O 2 로유발한산화적손상에대해농도의존적으로세포생존률을증가시켰다. 2. 금은화물추출물은 HaCaT keratinocyte 에서 H 2O 2 로유발한 DNA chromatin 의응축및 DNA 분절을감소시켰다. 3. 금은화물추출물은 HaCaT keratinocyte 에서 H 2O 2 로유발한 ROS의생성을농도의존적으로억제하였다. 이상의결과, 금은화물추출물은사람각질형성세포인 HaCaT keratinocyte에서 H 2O 2 로유발한산화적인스트레스에서세포보호및 ROS 생성억제효과가우수하게나타났으며, 이와같은결과를바탕으로금은화물추출물이피부의산화적손상을개선하는데주요하게작용할수있을것으로추측된다. 추후에금은화물추출물의피부에서산화적인손상을억제하는구체적인기전연구가추가적으로필요할것으로사료된다. Reference 1. Schools of Korean Medicine Textbook complolation committee. Bonchohak. Seoul : Yeonglimsa. 2007 : 240-1. 2. Ahn SH, Kim HH. Lonicerae Flos inhibited COX-2 and MMP-9 in LPS induced Arthritis of mouse through regulation of MIF. Kor J Ori Med Phywiol Pathol. 2010 ; 24(2) : 242-8. 3. Dan Bensky, Andrew Gamble. Chinese herbal medicins. Materia Media. WA : Eastland Press. 1986 : 85-6. 4. Huang KC. The Pharmacology of Chinese herbs (2nd Ed.). FL : CRC. 1998 : 8-9. 5. Bae JH, Kim MS, Kang EH. Antimicrobial Effect of Lonicerae Flos Extracts on Food-borne Pathogens. Food Sci Biotechnol. 2005 ; 37(4) : 642-7. 6. Joo JS, Kim JS, Jeong JG, Kim BK. Study of efficacy of Foeniculi fructus and Lonicerae flos extract on acute pancreatitis. Kor J Herbology. 2010 ; 25(4) : 39-45. 7. Ohata S, Sato N, Tu SH, Shinoda M, Protective effects of Taiwan crude drugs on experimental liver injuries. Yakugaku Zasshi. 1993 ; 113(12) : 870-80. 8. Rim BM, Rim CW, Choi JY. Chung YS, Jeong HG. Effects of Lonicera japonica extract as a biological

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